CN116497096A - Touchdown PCR detection method for detecting expression quantity of circular RNA - Google Patents
Touchdown PCR detection method for detecting expression quantity of circular RNA Download PDFInfo
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Abstract
The invention discloses a TouchDown PCR detection method for detecting the expression quantity of circular RNA, belonging to the field of molecular biology. The invention extracts total RNA in the HepG2 cell of the human liver cancer cell line, reversely transcribes the total RNA into cDNA, carries out real-time fluorescence quantitative detection on the circHDAC2 fragment by a TouchDown PCR program, discovers that the TouchDown PCR program can accurately distinguish the initial template amount, has the amplification efficiency of 97.90 percent, and can realize the relative quantification of the annular RNA. Compared with the amplification of common PCR amplification procedures, the invention has better specificity and sensitivity to the amplification of the circular RNA. The method reduces the difficulty of primer design, can efficiently detect the expression quantity of the annular RNA, and lays a foundation for the deep research of the annular RNA in the future.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a TouchDown PCR detection method for detecting the expression quantity of circular RNA.
Background
Circular RNA is a covalently closed circular RNA, and has the advantage of being free of exonuclease, unlike normal linear RNA. At present, the method has intensive researches on the aspects of regulating the occurrence and the development of diseases, treating and diagnosing and the like, and is a great hotspot of research in the field of epigenetic science. In recent years, there have also been studies on the application of circular RNAs to vaccine development. Circular RNAs have many special aspects in research methods due to their special circular structure, such as circular RNAs do not have a polyA tail, and therefore Oligo dT cannot be used as a reverse transcription primer; the circular RNA is of a circular structure, and a primer pair with a junction must be designed when PCR primer design is carried out so as to ensure the specificity of the product. The primer design method limits the selection range of primer pairs and increases the design difficulty. For this primer, it is difficult to find a suitable annealing temperature when amplification is performed using a conventional PCR procedure, and the product is likely to contain dimers, and the amplification conditions containing dimers cannot be used as real-time fluorescent quantitation. In addition, qRT-PCR generally requires higher primer design during operation to avoid non-specific products and to increase product specificity through higher annealing temperatures. However, when the abundance of the target gene in the sample is low, the target gene is difficult to detect and relatively quantitate even by using a real-time fluorescent quantitative PCR technology.
Touchdown RCR is a simple and rapid method to optimize PCR, increasing specificity, sensitivity and yield without lengthy optimization and/or redesigning primers. The Touchdown PCR employs an initial annealing temperature that is higher than the predicted melting temperature (Tm) of the primer used, and then gradually transitions to a lower, more relaxed annealing temperature during successive cycles. Any difference in Tm for correct and incorrect anneals will yield a doubling of the exponential advantage in each cycle. This method was originally developed to avoid the complex reaction optimization procedure performed to determine the optimal annealing temperature, and was additionally applied to determine the DNA sequence of peptides of known sequence. Touchdown PCR has been widely used in standard PCR protocols, including reverse transcriptase dependent PCR, and cDNA library generation and single nucleotide polymorphism screening; qualitative detection of low abundance genes, such as bacteria with low expression levels in milk or other biological samples, is also often performed by using the touch down PCR method, but the application of touch down RCR to real-time fluorescence quantification has been rarely studied.
Antitubercular drug liver injury (Anti-tuberculosis drug-induced liver injury, ADLI) refers to the pathological process caused by hepatotoxic injury due to drugs or their metabolites or allergies of the liver to drugs and their metabolites during the use of antitubercular drugs. Previous studies by the inventors have found that there are differences in the expression of circular RNAs in the cellular model of ADLI and in patient serum samples. Therefore, it is needed to provide a method capable of accurately and qualitatively and quantitatively detecting the circular RNA, so as to lay a foundation for deep research of the circular RNA in the future.
Disclosure of Invention
The invention aims to provide a Touchdown PCR detection method for detecting the expression quantity of circular RNA, so as to solve the problems in the prior art. The TouchDown PCR detection method can effectively amplify low-abundance annular RNA, is superior to the common PCR in sensitivity and specificity, has the amplification efficiency of 97.90 percent, and can improve the accuracy of differential expression quantitative analysis.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a method for detecting the expression quantity of circular RNA, which comprises the step of detecting the expression quantity of the circular RNA by adopting a TouchDown PCR amplification method.
Further, the nucleotide sequence of the circular RNA is shown as SEQ ID NO. 5.
Further, the primer pair for amplifying the circular RNA is shown as SEQ ID NO. 1-2.
Further, the method comprises the following steps:
s1, extracting total RNA of a sample to be detected;
s2, carrying out reverse transcription by taking the total RNA of the sample as a template to obtain cDNA;
s3, carrying out TouchDown PCR amplification by using the cDNA as a template and utilizing the primer pair;
s4, calculating the expression quantity of the circular RNA according to the Ct value.
Further, in step S3, the TouchDown PCR amplification reaction system is Mix 10. Mu.L, cDNA 2. Mu.L, forward primer 0.5. Mu.L, reverse primer 0.5. Mu.L, RNase Free ddH 2 O was replenished to 20. Mu.L.
Further, in step S3, the reaction condition of the Touchdown PCR amplification is 95 ℃ for 2min;95 ℃ for 30s,61-52 ℃ for 20s,4 cycles, each cycle being reduced by 3 ℃;95 ℃ for 30s,60 ℃ for 30s,35 cycles; 72℃for 5min.
The invention also provides a primer pair for detecting the expression quantity of the circular RNA, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO. 1-2; the nucleotide sequence of the circular RNA is shown as SEQ ID NO. 5.
The invention also provides application of the primer pair in detecting the expression quantity of the annular RNA, wherein the detection is to detect the expression quantity of the annular RNA by adopting a TouchDown PCR amplification method; the nucleotide sequence of the circular RNA is shown as SEQ ID NO. 5.
The invention discloses the following technical effects:
the invention uses the Touchdown PCR technology to amplify the circular RNA circHDAC2 (circBase ID: hsa_circ_ 0077692), uses the gradient diluted cDNA template to carry out real-time fluorescence quantitative detection, and discovers that the Touchdown PCR program can accurately distinguish the initial template quantity, and the Ct value is linearly related to the-log value of the template quantity 2 And the amplification efficiency reaches 0.9962 and 97.90%, and the relative quantification of the circular RNA can be realized.
The invention compares the amplification effect of the Touchdown PCR program with that of the common PCR program and the quantitative detection effect of the Touchdown PCR program, and detects the change of the relative expression quantity of the circHDAC2 before and after the drug treatment, thereby indicating that the Touchdown PCR method has good specificity and sensibility to the amplification of the circular RNA and can be used for qualitative and quantitative detection in a laboratory.
The method reduces the difficulty of primer design, can efficiently detect the expression quantity of the annular RNA, and lays a foundation for the deep research of the annular RNA in the future.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows amplification products of two PCR reaction procedures on the same circular RNA, where M: DNAmarker; s: a sample; r: RNA cleaving enzyme, 3 replicates;
FIG. 2 is a graph showing the results of a TouchDown PCR amplification procedure, wherein A: an amplification curve; b: dissolution profile; c: sequencing the amplified product;
FIG. 3 is a standard curve of a TouchDown PCR amplification procedure;
FIG. 4 is a gel electrophoresis of the products of TouchDown PCR after 5-fold gradient dilution of cDNA concentration, where M: DNAmarker;1-5: original cDNA concentration, 5 1 Double dilution of cDNA,5 2 Double dilution of cDNA,5 3 Double dilution of cDNA,5 4 Diluting cDNA by times;
FIG. 5 shows the relative expression level change of the circular RNA detected by the TouchDown PCR.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The main object of the present invention is to improve a PCR method for amplifying circular RNA. However, the factors affecting PCR are very large and are limited by the performance of the PCR apparatus, and are also dependent on the reaction system and the reaction conditions, wherein the design of the primers, the quality of cDNA, and Mg 2+ The concentration, pH of the buffer solution, circulation conditions and other factors influence the PCR result, wherein the annealing temperature is more important. The Touchdown PCR provides a simpler optimization method, and can lead specific amplification products to generate strong competitive consistency to nonspecific amplification products, thereby improving the specificity and efficiency of the PCR. In Touchdown PCR, the temperature selected for the annealing step is initially set to 5℃to 10℃higher than the calculated Tm of the primer. Annealing under high intensity conditions favors the formation of perfect primer-template hybridization. In subsequent cycles, the annealing temperature was gradually reduced by a small amount, such that at the end of PCR, the annealing temperature was 2℃to 5℃lower than the calculated Tm of the primer. Because the efficiency of binding the primer to the target fragment is much higher than that of the wrong fragment, the target sequence becomes PC after several geometric amplification cyclesThe main product of R.
Examples
1. Materials and methods
1.1 materials
Main reagents and materials: human hepatoma cell line HepG2 (doctor Biotechnology Co., ltd.);
PrimeScript TM RT reagent Kit (Shanghai Bioengineering Co., ltd.); SYBRgreen with anit-Tap PCRmix (Beijing polymeric Biotechnology Co., ltd.); isoniazid, rifampin, pyrazinamide (beijing claibao technologies limited).
The main instrument is as follows: fluorescent quantitative PCR instrument (ABI StepOne plus, siemens technologies Co.).
1.2 primer sequences
Designing a primer: the sequence of circHDAC2 (circBase ID: hsa_circ_ 0077692) was searched from the circBase library, the last 20 nucleotide sequences of the sequence were copied and spliced in front of the original sequence, and Primer pairs were designed using Primer Premier 5 software. The downstream primer may be selected according to the general principles of primer design, requiring that the upstream primer must cross the junction site of the circular RNA.
Primers for amplifying the circHDAC2 and reference GAPDH gene sequences are shown in Table 1, and the nucleotide sequence of the circular RNAcircHDAC2 is shown in SEQ ID NO. 5.
TABLE 1
SEQ ID NO.5：
AGGCCCCATAAAGCCACTGCCGAAGAAATGACAAAATATCACAGTGATGAGTATATCAAATTTCTACGGTCAATAAGACCAGATAACATGTCTGAGTATAGTAAGCAGATGCAGAGATTTAATGTTGGAGAAGATTGTCCAGTGTTTGATGGACTCTTTGAGTTTTGTCAGCTCTCAACTGGCGGTTCAGTTGCTGGAGCTGTGAAGTTAAACCGACAACAGACTGATATGGCTGTTAATTGGGCTGGAGGATTACATCATGCTAAGAAATCAGAAGCATCAGGATTCTGTTACGTTAATGATATTGTGCTTGCCATCCTTGAATTACTAAAGTATCATCAGAGAGTCTTATATATTGATATAGATATTCATCATGGTGATGGTGTTGAAGAAGCTTTTTATACAACAGATCGTGTAATGACGGTATCATTCCATAAATATGGGGAATACTTTCCTGGCACAGGAGACTTGAGG。
1.3 Experimental methods
1.3.1 cell culture and pharmaceutical liver injury cell model establishment
HepG2 cells were seeded in 6-well plates using DMEM medium containing 10% fetal calf serum, 37℃and 5% CO 2 Culturing until the cell density reaches 80%, and performing drug intervention. In the experimental group, three drugs of isoniazid, rifampicin and pyrazinamide are dissolved in DMSO, cells are subjected to combined contamination treatment, and the final concentration of the drug effect is as follows: 150 μg/mL+300 μg/mL+800 μg/mL while controlling the final DMSO concentration below 1%. After 24h of combined action of the three drugs, the medium was discarded, the cells were washed three times with PBS, and total RNA of the cells was extracted by adding 1ml of the trizol reagent. A blank control and a solvent control were set up for the experiment.
Extraction and detection of 1.3.2RNA
Total RNA extraction from cells was strictly in accordance with the kit instructions. 1ml of LTrilzol was added to each well, cells were gently scraped off and transferred well to RNase-free EP tubes and sonicated on ice for 30s. 0.2mL of chloroform was added to each 1mL of LTril, mixed well with shaking, allowed to stand on ice for 2min, and centrifuged at 12000rpm at 4℃for 15min. The water sample layer was aspirated and placed in a 1.5mL enzyme-free EP tube, 0.5mL isopropyl alcohol was added, mixed upside down (non-shake), placed on ice for 10min, centrifuged at 12000rpm for 10min at 4℃and the supernatant removed. 1mL of pre-chilled 75% ethanol (DEPC water configuration) was added, vortexed to thoroughly mix the sample, and centrifuged at 7500rpm at 4℃for 5min. After removing the supernatant and sufficiently air-drying, 10. Mu.L of EPC water was aspirated to dissolve RNA, and immediately after that, the RNA concentration was measured on ice.
1.3.3 RT-PCR experiments
After extracting total RNA in cells by Trizol method, primeScript was used TM Reverse transcription is performed by the RT reagent Kit: primeScript RT Master Mix 4. Mu.L Total RNA 500ng was added to the solution and the mixture was filled to 20. Mu.L with nucleic-Free Water. The conditions are 37 ℃ for 15min;85 ℃,6s; forever at 4 ℃.
RT-PCR detection of the cDNA after reverse transcription was performed using SYBRgreen with anit-Tap kit: mix 10. Mu.L, cDNA 2. Mu.L, forward primer (10. Mu.M) 0.5. Mu.L, reverse primer (10. Mu.M) 0.5. Mu.L, RNase Free ddH 2 O was replenished to 20. Mu.L.
The normal RT-PCR procedure was performed at 95℃for 2min, (95℃for 3s;60℃for 30s;72℃for 15 s) for 40 cycles, 72℃for 5min.
Touchdown PCR procedure: 95 ℃ for 2min; (95 ℃ C. 30s,61-52 ℃ C. 20 s) 4 cycles, each cycle being reduced by 3 ℃; (95 ℃ 30s,60 ℃ 30 s) 35 cycles; 72℃for 5min. Both PCR program reaction systems were 20. Mu.L. With GAPDH as internal reference for homogenization treatment, with 2 -ΔΔCt The relative expression level of the circular RNA was calculated by the method, wherein Δct=ct Purpose(s) -Ct Internal reference ，ΔΔCt＝ΔCt Contamination group -ΔCt Control group 。
1.3.4 electrophoresis
mu.L of PCR amplification product was subjected to 100V electrophoresis on a 1% agarose gel for 45min, and observed and analyzed by an UVP gel imaging system.
1.3.5 calculation of amplification efficiency
The cDNA after reverse transcription was taken and subjected to gradient dilution at 5-fold concentration, and 5 cDNA templates at different dilution fold were obtained, respectively. Templates at 5 concentrations were amplified using the Touchdown PCR program. And (3) calculating an amplification standard curve by taking the Ct value as an ordinate and taking the template quantity-log value as an abscissa. The slope of the curve is denoted k. Amplification efficiency= (10 -(1/k) -1)×100％。
1.3.6 statistical analysis
Statistical analysis was performed with SPSS16.0, using two independent sample t-test to compare the differences between the two groups, detection level α=0.05.
2. Results
2.1TouchDown PCR and ordinary PCR amplification of target Gene fragments
Since RNA-cleaving enzymes are capable of cleaving linear RNA, whereas circular RNA is retained due to the specific covalently closed circular structure, in this example the same sample that was subjected to RNA-cleaving enzyme treatment (RNase R enzyme, epicentre Corp.) and that was not was subjected to reverse transcription. The enzyme treatment method comprises the following steps: total RNA was digested at 37℃for 30min at a rate of 4U/. Mu.g RNA, and the enzyme was inactivated at 85℃for 5min after the completion of the digestion. RNA concentration was then calculated by detecting the absorbance of A260 and A280 and reverse transcription was performed using 500ng RNA.
Then, each sample was amplified by using two methods, ordinary RT-PCR and TouchDown PCR, and the results showed that under the same other conditions: 1) The abundance of RNA decreases after RNA shearing enzyme treatment; 2) The specificity of the Touchdown PCR procedure was significantly higher than that of the normal PCR procedure, and the specificity of the Touchdown PCR procedure was better in the different samples than in the normal PCR procedure (fig. 1). The upper part of the figure shows the amplification effect of the common RT-PCR program, and the lower part shows the amplification effect of the TouchDown PCR program under the same conditions of the same sample.
2.2 analysis of amplified products of the Touchdown PCR program
After gradient dilution of the cDNA template, circular RNA and GAPDH internal controls were amplified using the TouchDown PCR program, and single amplified products were found (FIG. 2A and FIG. 2B). And then sequencing the product of the circular RNA, and comparing the product with the circular RNA sequences in the circBase library, wherein the sequences of the circular RNA and the circular RNA are identical, so that the amplified product is correct. The partial sequencing results are shown in FIG. 2C.
2.3 amplification efficiency of Touchdown PCR program
The amplification efficiency of the TouchDown PCR procedure was calculated by gradient dilution of the template. 5-fold gradient concentration dilutions were performed with cDNA templates to obtain a total of 5 concentrations. Setting the initial template quantity to 5 5 After dilution, the mixture is sequentially 5 4 ，5 3 ，5 2 ，5 1 A scatter plot is made with its log value as the abscissa and the corresponding Ct value as the ordinate, as shown in fig. 3. Slope k= -3.3732, r 2 At 0.9962 (FIG. 3), it was suggested that the TouchDown PCR program could effectively distinguish between changes in the initial template amount, and that the Ct value was linearly related to the log of the template amount. According to the formula, the amplification efficiency= (10 (-1/-3.3732) -1)×100％＝97.90％。
2.4Touchdown PCR sensitivity analysis
Because the abundance of circular RNA in the sample is low, the invention uses cDNA template to dilute with 5 times gradient concentration, and carries out the Touchdown PCR program, the gel electrophoresis result shows that only one item of gene has no other nonspecific bands, the band definition degree is directly related to the initial template amount, and the Touchdown PCR program has good sensibility and relative quantification capability (figure 4).
2.5 relative quantification of circular RNA for TouchDown PCR program
After the cells are treated by the combination of the antitubercular drugs and the three drugs for 24 hours, total RNA is extracted, and the quantitative detection of qRT-PCR is carried out on the annular RNA by utilizing a TouchDown PCR program, so that the expression level of the annular RNAcircHDAC2 is obviously reduced after the drug treatment (model group) (figure 5). The method shows that the drug treatment influences the expression abundance of the annular RNA, and the expression quantity change of the annular RNA can be effectively detected by using a TouchDown PCR program.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A method for detecting the expression level of a circular RNA, comprising the step of detecting the expression level of the circular RNA by using a touchdown pcr amplification method.
2. The method of claim 1, wherein the nucleotide sequence of the circular RNA is set forth in SEQ ID No. 5.
3. The method according to claim 2, wherein the primer pair for amplifying the circular RNA is as shown in SEQ ID No. 1-2.
4. A method according to claim 3, comprising the steps of:
s1, extracting total RNA of a sample to be detected;
s2, carrying out reverse transcription by taking the total RNA of the sample as a template to obtain cDNA;
s3, carrying out TouchDown PCR amplification by using the cDNA as a template and utilizing the primer pair;
s4, calculating the expression quantity of the circular RNA according to the Ct value.
5. The method according to claim 4, wherein in step S3, the TouchDown PCR amplification reaction system is Mix 10. Mu.L, cDNA 2. Mu.L, forward primer 0.5. Mu.L, reverse primer 0.5. Mu.L, RNaseF primer ddH 2 O was replenished to 20. Mu.L.
6. The method according to claim 4, wherein in step S3, the reaction conditions for the TouchDown PCR amplification are 95℃for 2min;95 ℃ for 30s,61-52 ℃ for 20s,4 cycles, each cycle being reduced by 3 ℃;95 ℃ for 30s,60 ℃ for 30s,35 cycles; 72℃for 5min.
7. A primer pair for detecting the expression quantity of circular RNA, which is characterized in that the nucleotide sequence of the primer pair is shown as SEQ ID NO. 1-2; the nucleotide sequence of the circular RNA is shown as SEQ ID NO. 5.
8. The use of the primer pair according to claim 7 for detecting the expression level of a circular RNA, wherein the detection is to detect the expression level of the circular RNA by using a Touchdown PCR amplification method; the nucleotide sequence of the circular RNA is shown as SEQ ID NO. 5.
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